A method for analyzing DNA in the field of genetic engineering is a very useful means for searching for novel genes and pathogenic genes, and diagnosing genetic diseases, cancer diseases, infectious diseases and so on. As a method for amplification and analysis of a target gene, a polymerase chain reaction (PCR) method is widely used (U.S. Pat. Nos. 4,683,195, 4,683,202). As other means, there are a reverse transcriptase—polymerase chain reaction method (a RT-PCR method, Trends in Biotechnology, 10, 146-152, 1992), a ligase chain reaction method (LCR, EP 320308) and so on. Because these techniques involve steps such as strand separation of double-stranded DNA into single-strands, synthesis of complementary strands from primers, etc., requiring high- and low-temperature reactions to be repeated many times; therefore, a strict temperature controller is required, and a long time for temperature setting causes time losses. Further, the above techniques require an expensive enzyme.
Thereafter, a DNA amplification method under isothermal conditions was developed to solve the problem of temperature control. For example, there are a strand displacement amplification method (SDA, Nucleic Acid Res., 20, 1691–1696, 1992), a nucleic acid sequence based amplification method (NASBA, Nature, 350, 91–92, 1991) and a Qβ replicase method (BioTechnology, 6, 1197–1202, 1988). These methods can be advantageously carried out under isothermal conditions, but need expensive enzymes such as DNA polymerases, restriction endonucleases, etc.
These isothermal nucleic acid amplification methods have some problems in terms of the number of primers and operation, and it is demanded to establish a nucleic acid amplification method and a nucleic acid detection method of simple techniques at low cost without using enzymes.
On the other hand, the amplification of genes by the branched DNA probe method involves previously synthesizing a branched polymer single-stranded DNA probe and hybridizing it to a target gene to detect the target gene. However, the hybridization of the branched polymer single-stranded DNA probe to the target gene takes a long time because the branched DNA probe is a polymer. In addition, the branched polymer single-stranded DNA is limited in size, so that the detection of the target gene is also limited.
In view of the problems described above, the present applicant previously proposed a novel isothermal nucleic acid amplification method (a method for forming a probe-polymer) without using enzymes (EP 1002877A). This proposed method makes use of a pair of probes each composed of three portions (HoneyComb Probe, referred to hereinafter as HCP), and the three portions in the first probe are composed of base sequences complementary to each other, and the base sequences of both the probes are designed such that upon reaction, the three regions in one probe hybridize to only such regions in the other probe. By this method, a plurality of pairs of the probes upon reaction can hybridize to each other to form a polymer of the probes (a Probe Alternation Link Self-Assembly Reaction, referred to hereinafter as a PALSAR method).
This proposed method for forming a probe-polymer can be used to detect a target gene in a sample, and realizes an epoch-making, simple and inexpensive technique by isothermal operation without using enzymes.